Ultra-High Resolution Solid-State NMR for High Molecular Weight Proteins on GHz-Class Spectrometers

Ultra-High Resolution Solid-State NMR for High Molecular Weight Proteins on GHz-Class Spectrometers | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Ultra-High Resolution Solid-State NMR for High Molecular Weight Proteins on GHz-Class Spectrometers Songlin Wang , Thirupathi Ravula , John A. Stringer , Peter L. Gor’kov , Owen A. Warmuth , Christopher G. Williams , View ORCID Profile Alex F. Thome , View ORCID Profile Leonard J. Mueller , Chad M. Rienstra doi: https://doi.org/10.1101/2025.05.05.652283 Songlin Wang 1 National Magnetic Resonance Facility at Madison (NMRFAM), University of Wisconsin-Madison , WI, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: swang2256{at}wisc.edu crienstra{at}wisc.edu Thirupathi Ravula 2 Department of Biochemistry, University of Wisconsin-Madison , Madison, WI, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site John A. Stringer 5 Phoenix NMR , Loveland, CO, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Peter L. Gor’kov 6 National High Magnetic Field Laboratory (NHMFL), Florida State University , Tallahassee, FL, USA 7 Black Fox LLC , Tallahassee, FL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Owen A. Warmuth 2 Department of Biochemistry, University of Wisconsin-Madison , Madison, WI, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christopher G. Williams 8 Department of Chemistry, University of California-Riverside , Riverside, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alex F. Thome 8 Department of Chemistry, University of California-Riverside , Riverside, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alex F. Thome Leonard J. Mueller 8 Department of Chemistry, University of California-Riverside , Riverside, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Leonard J. Mueller Chad M. Rienstra 1 National Magnetic Resonance Facility at Madison (NMRFAM), University of Wisconsin-Madison , WI, USA 2 Department of Biochemistry, University of Wisconsin-Madison , Madison, WI, USA 3 Department of Chemistry, University of Wisconsin-Madison , Madison, WI, USA 4 Morgridge Institute for Research , Madison, WI, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: swang2256{at}wisc.edu crienstra{at}wisc.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract NMR spectroscopy is a powerful technique with broad impact across the physical and life sciences, and ultra-high field, GHz-class NMR spectrometers offer exceptional overall performance including superior resolution and sensitivity. While the resolution is fundamentally limited by molecular tumbling for solution NMR, solid-state NMR (SSNMR) is constrained only by instrumentation, making it well-suited for studying large and complex systems. To fully leverage UHF magnets for magic-angle-spinning SSNMR, it is essential to eliminate linebroadening arising from magnetic field drift and couplings among the nuclear spins. We address these challenges using an external 2 H lock to compensate the field drift and Long-Observation-Window Band-Selective Homonuclear Decoupling (LOW-BASHD) to suppress 13 C homonuclear couplings. We thereby achieve better than 0.2 ppm resolution in proteins up to 144 kDa, enabling unique site resolution for over 500 amide backbone pairs in 2D experiments. This exceeds the resolution available from solution NMR for large biological molecules, greatly expanding the potential of GHz-class NMR for research in life sciences. Teaser Ultra-high field NMR enables scientists to observe the finest details of large biomolecules, and this study overcomes key challenges to achieve a new benchmark for resolution in solid-state NMR of high molecular weight proteins. Introduction With advancements in superconducting technologies, nuclear magnetic resonance (NMR) has entered the GHz (10 9 Hz) era. Ultra-high field (UHF) NMR spectrometers now offer outstanding resolution and sensitivity, substantially facilitating research in materials and life sciences.( 1 - 4 ) High molecular weight biological systems, such as enzymes, assemblies, transporters, receptors, and motor proteins, particularly benefit from increased field strength, as the improved resolution enhances spectral dispersion and enables the isolation of signals of interest from overlapping resonance peaks. However, solution NMR faces inherently limitations in studying large biological molecules because its resolution is constrained by molecular tumbling rates. As molecular size increases, slower tumbling leads to severe line broadening, diminishing the resolution gains from UHF. It restricts routine solution NMR studies to proteins below 40 kDa, except in a few special cases.( 5 , 6 ) In contrast, for magic-angle-spinning (MAS) solid-state NMR (SSNMR), resolution continues to improve with increasing magnetic field strength, regardless of molecular size. As a result, SSNMR is particularly well-suited for studying large and complex biological systems and benefits the most from advancements in magnet technology. The development of high-field NMR has historically been constrained by magnet technology, with early instruments relying on resistive magnets that exhibited insufficient field homogeneity and temporal stability.( 7 ) A breakthrough occurred in the 1960s with the introduction of NbTi superconducting magnets, which enabled field strengths up to approximately 270 MHz (6.3 T). At that time, concerns were raised regarding the feasibility of higher fields due to limitations in probe design and radiofrequency (RF) performance. However, Hoult and Richards provided a critical theoretical analysis demonstrating that increased magnetic fields would enhance sensitivity and resolution, overcoming initial skepticism.( 8 ) Despite these advancements, NbTi-based magnets reached a practical limit of ∼400 MHz (9.4 T), leading to a decade-long stagnation in field progression.( 9 ) The emergence of Nb□Sn superconducting wire and improved cryogenic technology in the late 1980s enabled the development of 500 and 600 MHz systems, followed by 750 MHz magnets by the mid-1990s. Further refinements in Nb□Sn processing, combined with the use of 2 K cryogenic pumping, allowed for 800 and 900 MHz systems by the early 2000s.( 10 ) However, for nearly two decades, 900 MHz remained the practical upper limit of commercial NMR systems. A few specialized projects exceeded this threshold, such as the first GHz NMR (1.02 GHz) developed in Japan, and the 1.5 GHz Series Connected Hybrid (SCH) magnet at the National High Magnetic Field Laboratory (NHMFL), but these systems faced significant challenges, particularly regarding magnet stability and widespread accessibility.( 11 , 12 ) The advent of high-temperature superconducting (HTS) materials, such as YBCO, has now overcome this limitation, enabling commercial GHz-class NMR spectrometers with field strengths of 1.1 and 1.2 GHz since 2020. Even higher fields, including 1.3 GHz, are currently in development.( 13 , 14 ) According to Bruker Corporation, more than 20 GHz-class NMR spectrometers have been installed globally over the past five years, and the number continues to grow rapidly. GHz-class NMR spectrometers provide outstanding resolution and sensitivity; however, these systems face challenges related to magnetic field instability, including rapid drift, non-linear fluctuations, and field gradients that depend upon the magnet bore temperature. These issues are particularly problematic in newly energized magnets and can persist for months or even years after installation, as a consequence of the unique properties of the HTS technology.( 15 ) These instabilities degrade spectral quality and complicate the standardization of chemical shift referencing,( 16 ) resulting in greater uncertainty in peak positions and compromising one of the major benefits of utilizing higher magnetic fields. While the internal deuterium ( 2 H) lock technique has been widely employed in solution NMR to address field instability, its application in SSNMR has been limited, as internal locks cause degradation of the RF probe performance and many SSNMR samples lack 2 H nuclei with sufficiently narrow signals to use for the lock control. Although examples of effective external 2 H locks for MAS SSNMR have been demonstrated,( 17 , 18 ) SSNMR probes delivered along with the GHz-class magnets from Bruker lack this capability. One alternative approach utilizes simultaneous acquisition of reference spectra to reduce the variations when processing the data,( 19 ) although this method requires modifications to standard data collection and processing workflows and may be limited to samples containing a strong, narrow solvent signal. Therefore, many types of samples, such as membrane proteins, fibrils, drug formulations, and complexes, are not compatible with this strategy. In this study, we introduce SSNMR probes equipped with external 2 H lock coils compatible with the unique geometrical limitations of UHF HTS magnets. We achieve sufficient resolution for high molecular weight microcrystalline protein samples to resolve the fine structure arising from homonuclear 13 C- 13 C scalar couplings. We then apply Long-Observation-Window Band-Selective Homonuclear Decoupling (LOW-BASHD)( 20 ) to enhance the resolution and sensitivity by a factor of two. In combination, these techniques yield ultra-high resolution 13 C SSNMR spectra, with typical linewidths of 0.1-0.3 ppm for 13 C and 15 N in very large proteins, enabling the unique identification of the large majority of the 665 backbone amide pairs for a 144 kDa protein assembly in standard 2D spectra. Results Design of the SSNMR probe with external 2 H lock The design of a SSNMR probe with external 2 H lock requires that both the sample and lock coils lie within the limited range of the homogeneous magnetic field (“sweet spot”) in HTS magnets. Whereas conventional magnets typically have a sweet spot of at least 60 mm, this specification is not achieved in the UHF magnets. To image the field gradient for the 25.8 Tesla (1.1 GHz 1 H Larmor frequency) Bruker Ascend HTS magnet that was installed at the National Magnetic Resonance Facility at Madison (NMRFAM, University of Wisconsin-Madison) in late 2023, we varied the vertical position of a Phoenix 1.6 mm probe within the magnet, and acquired a series of 2 H 1D spectra from D 2 O packed in a 1.6 mm rotor with spinning at 5.8 kHz. During this measurement, all shim parameters were set to zero to visualize coil images, which became narrower as the sample approached the magnetic field center ( Fig. 1A ). The position defined as 0 mm corresponds to the highest probe position allowed by the top of the shim stack. The magnetic field center was determined to be 20 mm below this point, with an effective field range of approximately 35 mm. Compared with conventional superconducting magnets, this geometry is approximately a factor of two smaller in the Z direction, requiring a significantly more compact design to accommodate the high-power sample coil as well as the lock coil within the same magnetic field. Download figure Open in new tab Fig. 1. Mapping the effective field range and probe design with external 2 H lock. (A) mapping the effective field range of the 1.1 GHz NMR spectrometer at NMRFAM. The positions are defined as distances to the highest probe position allowed by the top of the shim stack. (B) SSNMR probes with external 2 H lock coil from PhoenixNMR (top) or Black Fox (bottom). Red dots indicate the centers of the sample coils, and red boxes highlight the 2 H lock coils. The numbers illustrate the distances from the center of the sample coil to the center of the lock coil. (C) Optimal shimming results for adamantane in the sample coil (top) and D 2 O in the 2 H lock coil (bottom) for the Black Fox 1.6 mm probe. Fig. 1B illustrates two probes with suitable external 2 H lock coils, incorporating epoxy sealed capillaries containing D 2 O as the lock sample. The vertical difference between the two coils is 12 mm for the Phoenix probe ( Fig. 1B , top) and 32 mm for the Black Fox probe ( Fig. 1B , bottom). To achieve the best shimming results for both coils, we detected 13 C signal from adamantane in the sample coil( 21 ) and 2 H signal from D 2 O in the lock coil, using the Bruker NEO console capability for simultaneous acquisition with multiple receivers and a dual acquisition pulse program. We then shimmed the consensus magnetic field using a combination of automated and manual adjustments.( 22 ) The Black Fox probe achieved linewidths of 2.6 Hz (10 ppb) for 13 C and 6.7 Hz (40 ppb) for 2 H ( Fig.1C ), with comparable performance observed for the Phoenix probe (Fig. S1). The probe heads utilize susceptibility matched materials and 3D modeling to symmetrize the components near the sample to achieve ppb-scale homogeneity,( 23 ) despite the high magnetic field. We determined that a stable lock signal could be obtained with 2 H linewidths as large as 20 Hz without noticeable splitting, with a best practice of 10 Hz or less. Post-installation field drift of GHz-class NMR and performance of the external 2 H lock Relative to conventional, metal alloy superconducting wire, the initial field drift rates for HTS magnets can be an order of magnitude larger and take months to stabilize to within specified performance.( 15 ) For instance, the magnetic field of the 1.1 GHz NMR spectrometer at NMRFAM drifted up rapidly after energization (50 Hz/hour) and reached a maximum approximately 6 months after installation ( Fig. 2A ). The drift then accelerated downward; after 10 months, the drift rate for 13 C was measured at -9.6 ppb/hour ( Fig. 2B , blue). This field drift could be compensated upon activating the 2 H lock, reducing the variations over an 8-hour period by almost two orders of magnitude, from almost 80 ppb to less than 2 ppb ( Fig. 2B , red). Although some fraction of this improvement may be achieved by a passive drift correction, this presumes a constant overall drift rate, which is not true for periods longer than few hours. Download figure Open in new tab Fig. 2. Magnetic field drift and the performance of external 2 H lock. (A) Long-term post-installation field drift of the 1.1 GHz NMR spectrometer at NMRFAM. (B) Field drifts over 8 hours monitored using the downfield peak of adamantane with 2 H lock on (red) and off (blue). The measurement was conducted ∼10 months after installation. (C) Comparison of 15 N/ 13 CO 2D correlation spectra of ToHo-1 β-lactamase acquired with 2 H lock on (red), and off (blue). The LOW-BASHD was applied to both experiments. (D) Evaluation of long-term 2 H lock performance using 13 C 1D spectra of ToHo-1 β-lactamase acquired over 7 days. To demonstrate the practical significance of the 2 H lock for GHz-class spectrometers, we collected 15 N/ 13 CO 2D correlation spectra over 14 hours for Toho-1 β-lactamase, a 29 kDa bacterial enzyme involved in antimicrobial resistance,( 24 ) with and without 2 H lock using the 1.1 GHz NMR ( Fig. 2C ). We observed severe peak shape distortion without the 2 H lock, with a notable shift in peak positions. These adverse effects are unacceptable for the acquisition of high-quality 2D or 3D datasets for macromolecules that may require several consecutive days of data collection. Uncertainties in peak positions also complicate procedures for resonance assignments, which are required for all subsequent stages of data analysis when studying structure and dynamics by NMR. In contrast, the 2 H lock improved the sensitivity and eliminated artifactual peak asymmetries and referencing offsets ( Fig. 2C and Table S1).( 25 , 26 ) The benefits of 2 H lock are compounded for longer experiments; for example, with lock on, spectra varied by less than 0.03 ppm over 7 days ( Fig. 2D ), a ∼50-fold reduction relative to the native drift of the magnet (∼1.6 ppm). Temperature effect on external 2 H lock Another unique and challenging feature of HTS magnets that we observed is that field stability is highly sensitive to temperature variations in the magnet bore. These variations can arise from changes in experimental temperature, sample replacement, or probe reinsertion. We observed that re-establishing thermal equilibrium within the probe-magnet system after such changes could take several hours, consistent with previously reported stabilization times for GHz-class NMR systems.( 27 ) Since all SSNMR probes thus far designed for these magnets require lowering the probe out of the magnet to change the sample, it is therefore essential to access the impact of temperature fluctuations on the performance of the external 2 H lock. We first monitored the shift in 13 C and 2 H peak upon changing the experimental temperature through the variable temperature (VT) unit of the spectrometer. For the Black Fox probe, 13 C and 2 H signals moved in opposite directions due to field gradient variation ( Fig. 3A ). This causes the magnitude of change in 13 C signal to be exacerbated due to temperature variations when the lock is engaged ( Fig. 3B ). We repeated the same measurement using CD 3 CN as the lock sample, which has an opposite and little response to temperature changes compared to D 2 O (Fig. S2). However, a similar drift trend was observed ( Fig. 3C and 3D ). This result indicates that the temperature-dependent peak position drift is predominantly driven by the inhomogeneous response of the field gradient of the HTS magnet to temperature changes. This effect is independent of the specific probe used, as a similar temperature effect was also observed for the Phoenix probe ( Fig. 3E and 3F ), though with a smaller amplitude and faster equilibrium due to the closer proximity of the coil to the magnetic field center. It is important to note that temperature-induced peak shifts are reversible once the thermal equilibrium is restored. Besides temperature, cryogen filling and powering the Bruker Smart Nitrogen Liquefier (BSNL) system on or off were also found to affect 2 H lock performance significantly (Fig. S3). Download figure Open in new tab Fig. 3. Temperature effect on 2 H lock performance. (A) Temperature effects on downfield peak of adamantane in the sample coil (blue) and 2 H peak of D 2 O in the lock coil (black) with 2 H lock off. (B) Temperature effects on downfield peak of adamantane with 2 H lock on. Measurements were conducted using the Black Fox 1.6 mm probe. The temperatures labeled in the figure represent the VT temperatures. (C) and (D), similar measurements as described in (A) and (B), but using CD 3 CN as lock solvent. (E) and (F), similar measurement as described in (A) and (B), but using Phoenix 1.6 mm probe. The VT temperature changes are virtualized by colors in the plots. The dashed lines in the right panels indicate that the chemical shift changes are reversible upon temperature changes. LOW-BASHD decoupling The maximum spectral resolution achievable in SSNMR is determined almost entirely by the intrinsic properties of the sample itself, and this is true both at moderate magnetic field and at field above 1 GHz.( 28 , 29 ) For well-ordered microcrystalline protein samples, this allows, for example, the observation of fine structure due to the homonuclear J -couplings. With our instrument, the J -couplings are the dominant source of inhomogeneous line broadening and effective limit to the overall linewidth. Homonuclear decoupling is therefore essential to realizing the highest achievable resolution. Here we demonstrate that UHF is compatible with state-of-the-art homonuclear decoupling during direct detection, allowing remarkable resolution in protein SSNMR. Specifically, we make use of the LOW-BASHD approach to decouple the ∼55 Hz J CαC’ splitting during 13 C detection.( 20 ) Fig. 4A shows a comparison of 15 N/ 13 CO 2D correlation spectra of tryptophan synthase, an αββα heterodimer with an effective molecular weight of 72 kDa per αβ unit,( 30 ) acquired with and without LOW-BASHD. The refocusing of J CαC’ during direct detection by LOW-BASHD results in an approximately 2-fold improvement in resolution and sensitivity ( Fig.4A , Table S2). With 18 hours of data acquisition, approximately 500 unique well-resolved resonance peaks are observed in the 2D spectrum, consistent with the reported number of detectable residues.( 30 ) Moreover, it substantially improves the alignment of resonance peaks in protein backbone assignments using 3D experiments ( Fig. 4B ). Download figure Open in new tab Fig. 4. The LOW-BASHD approach improves sensitivity and resolution in 13 CO detected experiments, facilitating the sequential assignments. (A) Comparison of 15 N/ 13 CO 2D correlation spectra of Tryptophan synthase acquired without LOW-BASHD (blue) and with LOW-BASHD (red). (B) Examples of 3D strip plots of tryptophan synthase for sequential assignments. The left plots contain NCACO (blue), NCOCA (green), and CANCO with LOW-BASHD (purple). For comparison, the right plots replace the NCACO to NCACO with LOW-BASHD (red). Middle panel compare the zoomed strips (highlighted in boxes) to illustrate the improved peak alignment achieved with LOW-BASHD. Discussion The rapid field drift of HTS magnets has become a significant challenge, limiting the application of UHF NMR to complex research systems. Currently, the most widely used method for field drift correction in the SSNMR community is the passive drift compensation feature offered by Bruker Topspin software, which applies a constant linear correction to the magnetic field. While effective for slow and stable drift, this approach is less reliable for HTS magnets, where drift is fast and inherently nonlinear over extended periods, leading to cumulative errors. In contrast, the external 2 H lock enables real-time correction of field fluctuations regardless of drift rate or linearity. The external 2 H lock for SSNMR was first introduced by Paulson and Zilm in 2008,( 18 ) using a spherical sample bulb filled with D 2 O solution placed above the probe and connected to a separate circuit from the top of the spectrometer. However, this design is incompatible with modern HTS-based GHz-class NMR spectrometers because of the short length of the sweet spot of the magnet, along with the design of the Bruker shim stack, which restricts the proximity of the lock coil to the sample coil. The probes presented in this study overcome these limitations by integrating the 2 H lock coil directly into the probe head, minimizing the distance between the lock and sample coils to ensure compatibility with the restricted field range of HTS magnets. The temperature-dependent shift of the 2 H lock signal indicates that the external 2 H lock cannot correct field disturbances caused by temperature or sample changes. Instead, the effect may be amplified due to temperature-dependent field gradient variations in the HTS magnet. In practice, 1D spectra should be collected regularly in the first few hours following a sample or temperature change to verify field stability. The time required to restore thermal equilibrium varies depending on the probe design. Probes with closer proximity between the sample and lock coils, and better thermal insulation design, tend to stabilize more quickly. For example, the Black Fox probe typically requires ∼6 hours to reach thermal equilibrium after temperature changes, whereas the Phoenix probe stabilizes within ∼2 hours. This difference is attributed to the proximity of the Phoenix lock coil to the magic-angle spinning stator, as well as a plastic coating layer inside the probe head cover that improves the thermal insulation. However, once thermal equilibrium is reached, the performance of the 2 H lock is consistent across different probes. It is also important to note that temperature-induced peak shifts are fully reversible once the field stabilizes. Therefore, the 2 H lock and spectral referencing must be conducted at a consistent temperature for upcoming research samples to obtain accurate chemical shift measurements. The effectiveness of LOW-BASHD is determined by the magnitude of the J -coupling relative to other contributions to the linewidth through the effective 13 C transverse relaxation time ( T’ 2 ). Consequently, the resolution enhancement from LOW-BASHD will be less pronounced for signals where linewidths are predominantly determined by other homogeneous and inhomogeneous contributions. This explains in part why LOW-BASHD approach, originally developed for SSNMR, has been used primarily for 13 C-detected experiments in protein solution NMR.( 31 ) To maximize the 13 C resolution enhancement offered by LOW-BASHD in MAS SSNMR, it is crucial to minimize other linebroading contributions, primarily 1 H/ 13 C heteronuclear dipolar couplings. This requires the use of high-power 1 H decoupling, which can lead to significant RF-induced sample heating when applied for extended durations. To mitigate the sample heating, the probes described in this study incorporate a dual resonator low electric field (“Low-E”) design, which effectively reduces the heat generation from the 1 H channel.( 32 , 33 ) This capability is particularly critical for 13 C detection with GHz-class NMR, where probe RF efficiency for 1 H is relatively low and signal lifetimes are prolonged. For example, on the 1.1 GHz NMR spectrometer, the 15 N and 13 C α T 2 relaxation times of the tryptophan synthase sample were measured at 23 ms and 9 ms, respectively. As a result, the total acquisition times for multi-dimensional experiments typically exceed 50 ms with high-power 1 H decoupling. The Low-E resonator design is therefore essential for enabling extended high-power 1 H decoupling while ensuring sample safety in 13 C detected experiments using UHF NMR. In summary, we introduced two SSNMR probes with distinct 2 H lock coil designs, demonstrating the critical role of 2 H lock in ensuring high-quality and accurate data acquisition on GHz-class NMR systems. The influence of temperature on field stability affects the 2 H lock performance, underscoring the importance of maintaining consistent experimental conditions. Additionally, the exceptional resolution offered by UHF NMR exposes the effect of 13 C- 13 C J -coupling splitting in 13 C-detected experiments. This issue is effectively resolved through the application of the LOW-BASHD approach, which not only eliminates splitting but also substantially enhances sensitivity and ensures precise resonance assignments. To demonstrate the power of these techniques, we showcased an ultra-high resolution 2D spectrum of the 144 kDa protein tryptophan synthase, achieving unique site resolution for several hundred residues. We envision that the application of external 2 H lock and LOW-BASHD in GHz-class NMR will open new opportunities in drug discovery, structural biology, and advanced materials research. Materials and Methods Instrumentation All NMR experiments were performed on a Bruker NEO 1.1 GHz spectrometer at NMRFAM. Two probes assessed in this study were the Black Fox 1.6 mm HCN triple-resonance probe (Black Fox LLC) and the Phoenix 1.6 mm HXY triple-resonance probe (PhoenixNMR LLC). Both probes featured external 2 H lock functionality, utilizing D 2 O sealed in a 1.5 mm OD × 12.5 mm long glass capillary (New Era) as lock sample. 20 mM Cu 2+ doped into D 2 O to accelerate the 2 H T 1 relaxation. The capillaries were flame-sealed at one end. D 2 O was added via centrifuge until full, the other end was then sealed with epoxy (J-B WELD). Bubbles need to be avoided to achieve good shim and lock results. For the Black Fox probe, the capillary was inserted horizontally into the lock coil and fixed using two 1.2×0.6 mm O-rings. For the Phoenix probe, the capillary was inserted vertically into the lock sample slot, and the insertion hole was covered with a small piece of tape for stabilization. For ToHo-1 β lactamase and tryptophan synthase samples, all spectra were collected at 25 kHz MAS rate, and the VT temperature was set as -5 ºC. All spectra were referenced to DSS, using adamantane as a secondary external standard. The downfield 13 C signal of adamantane was referenced to 40.48 ppm. Preparation of Toho-1 β-lactamase Uniformly labeled 13 C- 15 N Toho-1 was produced, purified, and crystallized following a modified version of a previously described protocol.( 24 , 34 ) In brief, BL21 E. coli cells transformed with a Toho-1 β-lactamase expression plasmid were overexpressed in 500 mL of kanamycin-supplemented minimal media containing 4.0 g 13 C-glucose, 3.0 g 15 N-NH 4 Cl, 200 mM Sorbitol, and 5 mM β-iene induced by 1 mM IPTG and grown overnight. Cells were harvested by centrifugation and resuspended in 20 mM MES, pH 6.5 (Buffer A) and lysed via sonication using a Branson 450D Digital Sonifier (Emerson Industrial Automation, St. Louis, MO, USA). Protein was purified through a 5 mL HiTrap SP Sepharose FF column (GE Healthcare, Pittsburgh, PA, USA) pre-equilibrated with Buffer A, and protein separation was achieved using a linear gradient with 20 mM MES, 300 mM NaCl, pH 6.5 (Buffer B) and protein elution monitored by UV absorbance. Collected protein fractions were pooled for size-exclusion chromatography using a 120 mL HiLoad Superdex S-200 column equilibrated with Buffer A. Elution was again tracked by UV absorbance, and the pooled protein fractions were concentrated using a 10 kDa MWCO Ultra Centrifugal Filter (Amicon) to a final concentration of 300 - 400 μM. Crystallization was initiated by mixing concentrated protein at a 1:1 ratio with crystal buffer (7 mM spermine, 30% PEG-8k) and incubated at 4 °C to allow crystallization over several days. Preparation of tryptophan synthase Uniformly labeled 13 C- 15 N Salmonelia typhimurium tryptophan synthase in E. coli was expressed and purified as previously described.( 35 , 36 ) Microcrystalline protein samples were prepared in 50 mM Cs-bicine, pH 7.8 crystal buffer containing 14% PEG-8000 and 3.0 mM spermine as previously described.( 35 ) Mapping effective magnetic field range of the 1.1 GHz spectrometer Field mapping was performed using the Phoenix 1.6 mm probe which was mounted on a probe frame attached to the Bruker shim stack, allowing free adjustment of the probe’s vertical position. A 1.6 mm rotor filled with D 2 O, spinning at 5.8 kHz, was used as the sample. All shim parameters were set to zero. The probe’s initial position was set to the highest allowable point, defined by the top of the shim stack. A 2 H 1D spectrum was collected at this position, showing a coil image. The probe was then lowered in 5 mm increments, with 2 H 1D spectra acquired at each step. The spectrum with the narrowest 2 H peak suggested that the sample was near the magnetic field center. The lower limit of the effective field range was determined when the 2 H signal became significantly broadened and undetectable. Shimming for double sample and 2 H lock coils simultaneously A dual-acquisition pulse sequence was employed to shim both coils simultaneously, combining a 13 C one-pulse experiment with low-power 1 H decoupling to acquire the 13 C signal from adamantane and a 2 H one-pulse experiment to detect the 2 H signal from D 2 O. The adamantane sample, packed in a 1.6mm Varian style rotor, was spun at 30 kHz. OPTO was used to evaluate the effect of each shim parameter on both 13 C and 2 H signals to determine which parameters significantly influence each coil.( 22 ) The information provided a guideline for manual adjustments. The 13 C signal from the sample coil was shimmed first to achieve the narrowest possible 13 C linewidth. Subsequently, shim parameters that had minimal impact on the sample coil but strongly affected the lock coil were adjusted to refine the 2 H signal. The objective was to achieve a 2 H linewidth below 20 Hz without noticeable splitting, while minimally compromising the 13 C linewidth. Once optimal shimming was achieved, the shim parameters were saved. For future shimming, only significant adjustments to the z value are typically required, while other parameters usually need minor fine-tuning only. Measuring 13 C and 2 H resonance peak drifting trajectories A 13 C one-pulse experiment with low-power 1 H decoupling was used to record the adamantane 13 C resonance peak drift. A 2.5µs 90º pulse was applied on 13 C channel, and signals were acquired with 15 kH continuous wave (CW) 1 H decoupling. The total acquisition time was 500 ms. To track 2 H signal drift, the lock function was turned off. A 2 H one-pulse experiment, using a 20 µs 90º pulse, was used to generate the 2 H signal from the lock channel, with an acquisition time of 500 ms. The 13 C and 2 H signals were acquired simultaneously using the same dual acquisition pulse program. The 1D spectra were recorded at 30 s intervals, and the data were stored in pseudo-2D containers for further processing. For the 13 C peak drifting measurement with 2 H lock, the same 13 C 1D spectra were acquired as described above except using a regular single 13 C acquisition pulse program. 2 H lock setup for research samples After completing the shimming process, the stability of the magnetic field was assessed by tracking the 13 C signal of adamantane with the 2 H lock activated. Once the 13 C peak position showed no significant drift, the field value was adjusted to position the left peak of adamantane at 40.48 ppm, ensuring an accurate reference to DSS. Sample changes do not require deactivating the 2 H lock, as the lock function automatically reactivates once the probe is back to its previous position in the magnet. Note that taking probe in and out may disturb the magnetic field, so additional time is needed for the field to stabilize following the sample change, typically 2 to 6 hours. During this period, pulse sequences can be optimized. For consistent and accurate chemical shift referencing for research samples, both the adamantane calibration and the experiments on research samples must be conducted at the same temperature. 15 N/ 13 CO 2D correlation spectra for ToHo-1 β lactamase and Tryptophan synthase The 2D NCO experiment was performed with the 13 C carrier frequency set at 175 ppm. The 15 N polarization was prepared by an adiabatic CP with a downward tangential ramp pulse on the 1 H channel, with a contact time of 2.0 ms, 15 N RF amplitude of 40 kHz, and an average 1 H RF amplitude of 60 kHz. 15 N polarization was then transferred to 13 CO using CP with an upward tangential ramp on the 13 C channel, with a contact time of 7.0 ms, 15 N RF amplitude of 10 kHz, and an average 13 C RF amplitude of 16 kHz. A CW decoupling of 1 H at 100 kHz was applied during this CP period. The t 1 acquisition time was 41.0 ms with a 40 µs increment for 1024 complex points. A 5.2 µs 13 C π-pulse was applied at the center of the t 1 period to decouple the J 15N-13C . The t 2 acquisition time was 30.7 ms with a 5 µs dwell time for 3072 complex points. SPINAL-64 1 H decoupling at 100 kHz was applied during acquisition. For LOW-BASHD experiments, LOW-BASHD detection was implemented in the direct dimension to decouple the 1 J C α -C’ using τ Dec = 3.2 ms and 72.5 µs cosine modulated Gaussian π-pulses, modulated at 37 kHz.( 20 ) The recycle delay was 1.5 s. The total experimental time was 14 hours. 15 N/ 13 C α / 13 CO 3D correlation spectrum (NCACO) for Tryptophan synthase The 3D NCACO experiment was performed with the 13 C carrier frequency set at 55 ppm. The 15 N polarization was prepared by an adiabatic CP with a downward tangential ramp pulse on the 1 H channel, with a contact time of 1.5 ms, 15 N RF amplitude of 40 kHz, and an average 1 H RF amplitude of 59 kHz. 15 N polarization was then transferred to 13 C α using CP with an upward tangential ramp on the 13 C channel, with a contact time of 7.0 ms, 15 N RF amplitude of 10 kHz, and an average 13 C RF amplitude of 15 kHz. A CW decoupling of 1 H at 100 kHz was applied during this CP period. The 13 C α / 13 CO polarization transfer was achieved using a 100.0 ms CORD mixing period with a 1 H RF amplitude of 25 kHz.( 37 ) The t 1 acquisition time was 15.4 ms with a 120 µs increment for 128 complex points. A 5.2 µs 13 C π-pulse was applied at the center of the t 1 period to decouple the J 15N-13C . The t 2 acquisition time was 6.4 ms with an 80 µs increment for 80 complex points. A 5.2 µs 13 C hard π-pulse, a 300 µs 13 C soft π-pulse with RSNOB shape at 55 ppm, and a 14.8 µs 15 N π-pulse were applied at the center of the t 2 period to decouple the J 13C - 13CX and J 13C α -15N . The t 3 acquisition time was 30.7 ms with a 5 µs dwell time for 3072 complex points. SPINAL-64 1 H decoupling at 100 kHz was applied during acquisition. For LOW-BASHD version of NCACO experiment, LOW-BASHD was implemented in the direct dimension to decouple the 1 J C α -C’ using τ dec =3.2 ms and 72.5 µs cosine modulated Gaussian π-pulses, modulated at 37 kHz. The recycle delay was 1.5 s. The 3D spectrum was acquired using a 25% NUS schedule and the total experimental time was 113.4 hours. 15 N/ 13 CO/ 13 C α 3D correlation spectrum (NCOCA) for Tryptophan synthase The 3D NCOCA experiment was performed with the 13 C carrier frequency set at 175 ppm. The 1 H to 15 N CP and the 15 N to 13 CO CP transfer conditions were identical to the 2D NCO experiment as described above. The 13 CO α / 13 C α polarization transfer was achieved using a 100.0 ms CORD mixing period with a 1 H RF amplitude of 25 kHz. The t 1 acquisition time was 15.4 ms with a 120 µs increment for 128 complex points. A 5.2 µs 13 C π-pulse was applied at the center of the t 1 period to decouple the J 15N-13C . The t 2 acquisition time was 7.7 ms with a 160 µs increment for 48 complex points. A 5.2 µs 13 C hard π-pulse, a 300 µs 13 C soft π-pulse with Rsnob shape at 175 ppm, and a 14.8 µs 15 N π-pulse were applied at the center of the t 2 period to decouple the J 13CO-13CX and J 13CO-15N . The t 3 acquisition time was 30.7 ms with a 5 µs dwell time for 3072 complex points. SPINAL-64 1 H decoupling at 100 kHz was applied during acquisition. The recycle delay was 1.5 s. The 3D spectrum was acquired using a 25% NUS schedule and the total experimental time was 136.2 hours. 13 C α / 15 N/ 13 CO 3D correlation spectrum (CANCO) for Tryptophan synthase The 3D CANCO experiment was performed with the 13 C carrier frequency set at 175 ppm. 13 C α polarization was prepared via adiabatic CP using a downward tangential ramp pulse on the 1 H channel, with the 13 C frequency adjusted to 55 ppm for 1 H- 13 C α transfer. The CP contact time was 1.5 ms, 13 C RF amplitude was 108 kHz, and the average 1 H RF amplitude was 82 kHz. Polarization was then transferred from 13 C α to 15 N using a CP with an upward tangential ramp on the 13 C channel. The contact time was 7.0 ms, 15 N RF amplitude was 10 kHz, and the average 13 C RF amplitude was 16 kHz. A 100 kHz 1 H CW decoupling was applied during this CP period. The 15 N polarization was transferred to 13 CO using CP with an upward tangential ramp on the 13 C channel. The 13 C frequency was changed back to 175 ppm before this CP. The contact time was 7.0 ms, with a 15 N RF amplitude of 10 kHz, and an average 13 C RF amplitude of 17 kHz. A 100 kHz 1 H CW decoupling was applied during this CP period. The t 1 acquisition time was 6.4 ms with an 80 µs increment for 80 complex points. A 5.2 µs 13 C hard π-pulse and a 300 µs 13 C soft π-pulse with Rsnob shape at 55 ppm were applied at the center of the t 1 period to decouple the J C α - CX . The t 2 acquisition time was 15.4 ms with a 120 µs increment for 128 complex points. A 5.2 µs 13 C π-pulse was applied at the center of the t 2 period to decouple the J N-C . The t 3 acquisition time was 30.7 ms with a 10 µs dwell time for 1536 complex points. A SPINAL-64 1 H decoupling at 100 kHz was used during acquisition. Additionally, LOW-BASHD detection was implemented in the direct dimension to decouple the J C α -CO using τ dec =3.2 ms and 72.5 µs Gaussian π-pulses, modulated at 37 kHz. The recycle delay was 1.5 s. The 3D spectrum was acquired using a 25% NUS schedule and the total experimental time was 107.4 hours. Funding This study made use of the National Magnetic Resonance Facility at Madison (NMRFAM), an NIH Biomedical Technology Development and Dissemination Center (P41GM136463). The 1.1 GHz NMR spectrometer was funded by the United States National Science Foundation (NSF) Mid-Scale Research Infrastructure Big Idea (1946970). Helium recovery equipment, computers, and infrastructure for data archive were funded by the University of Wisconsin-Madison, NIH (P41GM136463, R24GM141526), and NSF (1946970). L.J.M. was supported by the NIH (R01GM137008 and R35GM145369). Author contributions Conceptualization: SW, CMR. Methodology: SW, TR, JAS, PLG, CMR. Investigation: SW, TR, CGW, OAW, AFT, LJM, CMR. Visualization: SW. Supervision: CMR, LJM. Writing—original draft: SW, CMR, LJM. Writing—review & editing: SW, TR, JAS, PLG, OAW, CGW, AFT, LJM, CMR Competing interests J.A.S. is employed by Phoenix NMR, and P.L.G. is the founder of Black Fox LLC, both of which design and sell SSNMR probes with external 2 H lock functionality. All other authors declare they have no competing interests. Data and materials availability All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Information. The raw NMR data is available on the website of Network of Advanced NMR. The pulse sequences with LOW-BASHD decoupling were developed for the Bruker NEO console using Topspin 4.4.0. The pulse programs are available upon request. Supplementary Materials Acknowledgments Funder Information Declared National Institutes of Health, https://ror.org/01cwqze88 , P41GM136463 , R24GM141526 , R01GM137008 , R35GM145369 National Science Foundation, https://ror.org/021nxhr62 , 1946970 References 1. ↵ T. Le Marchand , T. Schubeis , M. Bonaccorsi , P. Paluch , D. Lalli , A. J. Pell , L. B. Andreas , K. Jaudzems , J. Stanek , G. Pintacuda , 1H-Detected Biomolecular NMR under Fast Magic-Angle Spinning . Chem. Rev . 122 , 9943 – 10018 ( 2022 ). OpenUrl CrossRef PubMed 2. M. Callon , D. Luder , A. A. Malar , T. Wiegand , V. Rimal , L. Lecoq , A. Bockmann , A. Samoson , B. H. Meier , High and fast: NMR protein-proton side-chain assignments at 160 kHz and 1.2 GHz . Chem Sci 14 , 10824 – 10834 ( 2023 ). OpenUrl CrossRef PubMed 3. F. A. Perras , A. L. Paterson , J. Reedijk , K. R. Poeppelmeier , “ 9.07 - High field solid-state nmr of challenging nuclei in inorganic systems ” in Comprehensive Inorganic Chemistry III ( Elsevier, Oxford , 2023 ), pp. 138 – 177 . 4. ↵ D. L. Bryce , G. A. Webb , “ Chapter One - Solid-state NMR of quadrupolar nuclei: Selected new methods and applications ” in Annual Reports on NMR Spectroscopy ( cademic Press , 2022 ), vol. 107 , pp. 1 – 46 . OpenUrl CrossRef 5. ↵ D. Bell , F. Lindemann , L. Gerland , H. Aucharova , A. Klein , D. Friedrich , M. Hiller , K. Grohe , T. Meier , B. van Rossum , A. Diehl , J. Hughes , L. J. Mueller , R. Linser , A.-F. Miller , H. Oschkinat , Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR – practical aspects . Journal of Biomolecular NMR 78 , 179 – 192 ( 2024 ). OpenUrl CrossRef PubMed 6. ↵ N. Bolik-Coulon , A. I. M. Sever , R. W. Harkness , J. M. Aramini , Y. Toyama , D. F. Hansen , L. E. Kay , Less is more: A simple methyl-TROSY based pulse scheme offers improved sensitivity in applications to high molecular weight complexes . Journal of Magnetic Resonance 346 , 107326 ( 2023 ). OpenUrl CrossRef PubMed 7. ↵ Z. Gan , H. T. Kwak , M. Bird , T. Cross , P. Gor’kov , W. Brey , K. Shetty , High-field NMR using resistive and hybrid magnets . J Magn Reson 191 , 135 – 140 ( 2008 ). OpenUrl CrossRef PubMed 8. ↵ D. I. Hoult , R. E. Richards , The signal-to-noise ratio of the nuclear magnetic resonance experiment . Journal of Magnetic Resonance (1969) 24 , 71 – 85 ( 1976 ). OpenUrl CrossRef Web of Science 9. ↵ A. Godeke , D. Cheng , D. R. Dietderich , P. Ferracin , S. O. Prestemon , G. Sabbi , R. M. Scanlan , Limits of NbTi and Nb 3 Sn, and Development of W&R Bi–2212 High Field Accelerator Magnets . IEEE Transactions on Applied Superconductivity 17 , 1149 – 1152 ( 2007 ). OpenUrl CrossRef 10. ↵ R. Fu , W. W. Brey , K. Shetty , P. Gor’kov , S. Saha , J. R. Long , S. C. Grant , E. Y. Chekmenev , J. Hu , Z. Gan , M. Sharma , F. Zhang , T. M. Logan , R. Bruschweller , A. Edison , A. Blue , I. R. Dixon , W. D. Markiewicz , T. A. Cross , Ultra-wide bore 900 MHz high-resolution NMR at the National High Magnetic Field Laboratory . J Magn Reson 177 , 1 – 8 ( 2005 ). OpenUrl CrossRef PubMed 11. ↵ K. Hashi , S. Ohki , S. Matsumoto , G. Nishijima , A. Goto , K. Deguchi , K. Yamada , T. Noguchi , S. Sakai , M. Takahashi , Y. Yanagisawa , S. Iguchi , T. Yamazaki , H. Maeda , R. Tanaka , T. Nemoto , H. Suematsu , T. Miki , K. Saito , T. Shimizu , Achievement of 1020MHz NMR . J Magn Reson 256 , 30 – 33 ( 2015 ). OpenUrl CrossRef PubMed 12. ↵ Z. Gan , I. Hung , X. Wang , J. Paulino , G. Wu , I. M. Litvak , P. L. Gor’kov , W. W. Brey , P. Lendi , J. L. Schiano , M. D. Bird , I. R. Dixon , J. Toth , G. S. Boebinger , T. A. Cross , NMR spectroscopy up to 35.2T using a series-connected hybrid magnet . J Magn Reson 284 , 125 – 136 ( 2017 ). OpenUrl CrossRef PubMed 13. ↵ J. Bascunan , S. Hahn , D. K. Park , Y. Iwasa , A 1.3-GHz LTS/HTS NMR Magnet-A Progress Report . IEEE Trans Appl Supercond 21 , 2092 – 2095 ( 2011 ). OpenUrl CrossRef PubMed 14. ↵ H. Maeda , J. Shimoyama , Y. Yanagisawa , Y. Ishii , M. Tomita , The MIRAI Program and the New Super-High Field NMR Initiative and Its Relevance to the Development of Superconducting Joints in Japan . Ieee Transactions on Applied Superconductivity 29 , 1 – 9 ( 2019 ). OpenUrl CrossRef 15. ↵ E. Nimerovsky , K. T. Movellan , X. C. Zhang , M. C. Forster , E. Najbauer , K. Xue , R. Dervisoglu , K. Giller , C. Griesinger , S. Becker , L. B. Andreas , Proton Detected Solid-State NMR of Membrane Proteins at 28 Tesla (1.2 GHz) and 100 kHz Magic-Angle Spinning . Biomolecules 11 , 752 ( 2021 ). OpenUrl CrossRef PubMed 16. ↵ J. L. Markley , A. Bax , Y. Arata , C. W. Hilbers , R. Kaptein , B. D. Sykes , P. E. Wright , K. Wuthrich , Recommendations for the presentation of NMR structures of proteins and nucleic acids. IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy . J Biomol NMR 12 , 1 – 23 ( 1998 ). OpenUrl CrossRef PubMed Web of Science 17. ↵ M. Takahashi , Y. Ebisawa , K. Tennmei , Y. Yanagisawa , M. Hosono , K. Takasugi , T. Hase , T. Miyazaki , T. Fujito , H. Nakagome , T. Kiyoshi , T. Yamazaki , H. Maeda , Towards a beyond 1 GHz solid-state nuclear magnetic resonance: external lock operation in an external current mode for a 500 MHz nuclear magnetic resonance . Rev Sci Instrum 83 , 105110 ( 2012 ). OpenUrl CrossRef PubMed 18. ↵ E. K. Paulson , K. W. Zilm , External field-frequency lock probe for high resolution solid state NMR . Review of Scientific Instruments 76 , ( 2005 ). 19. ↵ V. Rimal , M. Callon , A. A. Malar , R. Cadalbert , A. Torosyan , T. Wiegand , M. Ernst , A. Bockmann , B. H. Meier , Correction of field instabilities in biomolecular solid-state NMR by simultaneous acquisition of a frequency reference . Magn Reson (Gott) 3 , 15 – 26 ( 2022 ). OpenUrl CrossRef PubMed 20. ↵ J. O. Struppe , C. Yang , Y. Wang , R. V. Hernandez , L. M. Shamansky , L. J. Mueller , Long-observation-window band-selective homonuclear decoupling: increased sensitivity and resolution in solid-state NMR spectroscopy of proteins . J Magn Reson 236 , 89 – 94 ( 2013 ). OpenUrl CrossRef PubMed 21. ↵ C. R. Morcombe , K. W. Zilm , Chemical shift referencing in MAS solid state NMR . J Magn Reson 162 , 479 – 486 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 22. ↵ C. G. Borcik , B. DeZonia , T. Ravula , B. D. Harding , R. Garg , C. M. Rienstra , OPTO: Automated Optimization for Solid-State NMR Spectroscopy . J Am Chem Soc 147 , 3293 – 3303 ( 2025 ). OpenUrl CrossRef PubMed 23. ↵ J. Schonzart , R. Han , T. Gennett , C. M. Rienstra , J. A. Stringer , Magnetic Susceptibility Modeling of Magic-Angle Spinning Modules for Part Per Billion Scale Field Homogeneity . J Magn Reson 364 , 107704 ( 2024 ). OpenUrl CrossRef PubMed 24. ↵ V. V. Sakhrani , R. K. Ghosh , E. Hilario , K. L. Weiss , L. Coates , L. J. Mueller , Toho-1 beta-lactamase: backbone chemical shift assignments and changes in dynamics upon binding with avibactam . J Biomol NMR 75 , 303 – 318 ( 2021 ). OpenUrl CrossRef PubMed 25. ↵ J. C. C. Chan , R. Tycko , Recoupling of chemical shift anisotropies in solid-state NMR under high-speed magic-angle spinning and in uniformly C13-labeled systems . The Journal of Chemical Physics 118 , 8378 – 8389 ( 2003 ). OpenUrl CrossRef 26. ↵ M. J. Duer , G. A. Webb , “Solid-State NMR Studies of Molecular Motion” ( Academic Press , 2006 ), vol. 59 , pp. 41 – 116 . OpenUrl 27. ↵ A. A. Malar , L. A. Volker , R. Cadalbert , L. Lecoq , M. Ernst , A. Bockmann , B. H. Meier , T. Wiegand , Temperature-Dependent Solid-State NMR Proton Chemical-Shift Values and Hydrogen Bonding . J Phys Chem B 125 , 6222 – 6230 ( 2021 ). OpenUrl 28. ↵ G. De Paepe , N. Giraud , A. Lesage , P. Hodgkinson , A. Bockmann , L. Emsley , Transverse dephasing optimized solid-state NMR spectroscopy . J Am Chem Soc 125 , 13938 – 13939 ( 2003 ). OpenUrl CrossRef PubMed 29. ↵ R. W. Martin , E. K. Paulson , K. W. Zilm , Design of a triple resonance magic angle sample spinning probe for high field solid state nuclear magnetic resonance . Review of Scientific Instruments 74 , 3045 – 3061 ( 2003 ). OpenUrl CrossRef 30. ↵ A. Klein , P. Rovo , V. V. Sakhrani , Y. Wang , J. B. Holmes , V. Liu , P. Skowronek , L. Kukuk , S. K. Vasa , P. Guntert , L. J. Mueller , R. Linser , Atomic-resolution chemical characterization of (2x)72-kDa tryptophan synthase via four- and five-dimensional (1)H-detected solid-state NMR . Proc Natl Acad Sci U S A 119 , e2114690119 ( 2022 ). OpenUrl Abstract / FREE Full Text 31. ↵ J. Ying , J. Roche , A. Bax , Homonuclear decoupling for enhancing resolution and sensitivity in NOE and RDC measurements of peptides and proteins . J Magn Reson 241 , 97 – 102 ( 2014 ). OpenUrl CrossRef PubMed 32. ↵ S. A. McNeill , P. L. Gor’kov , K. Shetty , W. W. Brey , J. R. Long , A low-E magic angle spinning probe for biological solid state NMR at 750MHz . 197 , 135 – 144 ( 2009 ). OpenUrl 33. ↵ J. A. Stringer , C. E. Bronnimann , C. G. Mullen , D. H. Zhou , S. A. Stellfox , Y. Li , E. H. Williams , C. M. Rienstra , Reduction of RF-induced sample heating with a scroll coil resonator structure for solid-state NMR probes . J Magn Reson 173 , 40 – 48 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 34. ↵ S. J. Tomanicek , R. F. Standaert , K. L. Weiss , A. Ostermann , T. E. Schrader , J. D. Ng , L. Coates , Neutron and X-ray crystal structures of a perdeuterated enzyme inhibitor complex reveal the catalytic proton network of the Toho-1 beta-lactamase for the acylation reaction . J Biol Chem 288 , 4715 – 4722 ( 2013 ). OpenUrl Abstract / FREE Full Text 35. ↵ V. Copie , W. S. Faraci , C. T. Walsh , R. G. Griffin , Inhibition of alanine racemase by alanine phosphonate: detection of an imine linkage to pyridoxal 5’-phosphate in the enzyme-inhibitor complex by solid-state nitrogen-15 nuclear magnetic resonance . Biochemistry 27 , 4966 – 4970 ( 1988 ). OpenUrl CrossRef PubMed 36. ↵ S. A. Ahmed , C. C. Hyde , G. Thomas , E. W. Miles , Microcrystals of tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium are catalytically active . Biochemistry 26 , 5492 – 5498 ( 1987 ). OpenUrl CrossRef PubMed 37. ↵ G. Hou , S. Yan , J. Trebosc , J. P. Amoureux , T. Polenova , Broadband homonuclear correlation spectroscopy driven by combined R2(n)(v) sequences under fast magic angle spinning for NMR structural analysis of organic and biological solids . J Magn Reson 232 , 18 – 30 ( 2013 ). OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted May 06, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Ultra-High Resolution Solid-State NMR for High Molecular Weight Proteins on GHz-Class Spectrometers Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Ultra-High Resolution Solid-State NMR for High Molecular Weight Proteins on GHz-Class Spectrometers Songlin Wang , Thirupathi Ravula , John A. Stringer , Peter L. Gor’kov , Owen A. Warmuth , Christopher G. Williams , Alex F. Thome , Leonard J. Mueller , Chad M. Rienstra bioRxiv 2025.05.05.652283; doi: https://doi.org/10.1101/2025.05.05.652283 Share This Article: Copy Citation Tools Ultra-High Resolution Solid-State NMR for High Molecular Weight Proteins on GHz-Class Spectrometers Songlin Wang , Thirupathi Ravula , John A. Stringer , Peter L. Gor’kov , Owen A. Warmuth , Christopher G. Williams , Alex F. Thome , Leonard J. Mueller , Chad M. Rienstra bioRxiv 2025.05.05.652283; doi: https://doi.org/10.1101/2025.05.05.652283 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Biophysics Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17708) Bioengineering (13904) Bioinformatics (41992) Biophysics (21466) Cancer Biology (18618) Cell Biology (25531) Clinical Trials (138) Developmental Biology (13387) Ecology (19924) Epidemiology (2067) Evolutionary Biology (24337) Genetics (15615) Genomics (22521) Immunology (17749) Microbiology (40424) Molecular Biology (17194) Neuroscience (88673) Paleontology (667) Pathology (2839) Pharmacology and Toxicology (4827) Physiology (7650) Plant Biology (15160) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9826) Zoology (2271)